(1) Field of the Invention
The present invention relates to compounds used to label biomolecules for diagnostic and therapeutic purposes. In particular, it relates to fluorescent, chromophoric, pro-fluorescent and pro-chromophoric compounds that may be conjugated to biomolecules such as proteins and nucleic acids. Such compounds may be incorporated into linkers that may be used to link a ligand to a biomolecular probe allowing quantitation of the ligand bound to that molecular probe.
(2) Description of Related Art
Methods to detect interactions between biomolecules continues to be an area of active research as new and more sensitive methods are required to increase sensitivity, reduce costs and enable new detection methods. One of the most widely used methods is to directly label a biomolecule with a fluorescent molecule that fluoresces at a desired frequency. For example, a fluorescent molecule is modified with a thiol- or amino-reactive moiety such as succinimidyl esters or maleimides that form a covalent bound in the presence of a sulhydryl or amine group of a desired protein. The modified fluorescent molecule is isolated and reacted with the desired protein. The fluorescently labeled protein is then used to detect a desired target by monitoring the unique fluorescent frequency of the fluorophore. A variety of fluorophores have been modified with these moieties including fluorescein, rhodamine, Texas Red and cyanine dyes, Cy3 and Cy5. Unfortunately, the conjugation methods often cause quenching and photobleaching of the fluorophore and there can be interference with the observed signal if the unbound labeled biomolecule is not removed from the reaction mixture.
Other biolmolecules such as nucleic acids such as DNA, RNA, polynucleotide and oligonucleotides have been labeled with fluorophores is commonly accomplished by incorporating a fluorophore on the base moiety of a nucleoside triphosphate. These fluorescently labeled triphosphates are added to the polymerase chain reaction (PCR) or reverse transcription reaction wherein the labeled nucleoside is incorporated in the amplicon yielding a fluorescently labeled polynucleotide. These fluorescently labeled polynucleotides are probed using oligonucleotide microarrays identifying sequences present in the target. Unfortunately, the fluorophores used for labeling these biomolecules are not often stable to these synthesis conditions. In addition, the long-term stability of conjugates are low due to photobleaching, consequently, retention of the fluorescent signal is difficult when archiving microarrays.
A variety of references cite the use of fluorescent hydrazides, thiosemicarbazides and hydrazides to react with aldehydes on biological molecules for the detection of the aldehydes. For example Ahn et al. (B. Ahn, S. G. Rhee and E. R. Stadtman, Anal. Biochem. 161:245 (1987) describe the use of fluorescein hydrazide and fluorescein thiosemicarbazide for the fluorometric determination of protein carbonyl groups and for the detection of oxidized proteins on polyacrylamide gels. Proudnikov and Mirzabekov (Nucl. Acids Res. 24:4535 (1996)) describe labeling of DNA and RNA to identify acid-induced depurination that results in production of aldehyde moieties detected by reaction of fluorescent labels containing hydrazide groups in the presence of sodium cyanoborohydride. Others have labeled the reducing end of polysaccharides with fluorescent hydrazides. These methods are used to detect aliphatic aldehyde groups on biomolecules. In each of the references the fluorescent moiety is incorporated on the hydrazine or hydrazide that forms a hydrazone on reaction with the aldehyde present on the biomolecule.
It has been documented that hydrazones formed between certain aromatic aldehydes and aromatic hydrazines and not aromatic hydrazides or aromatic thiosemicarbazides form fluorescent molecules (J. Wong and F. Bruscato, Tet. Lett. 4593, 1968). It has also been reported that hydrazones formed specifically from 2-substituted aldehyde heterocycles and 2-substituted hydrazine heterocycles become fluorescent on chelation to zinc (D. E. Ryan, F. Snape and M. Winpe, Anal. Chim. Acta 58:101, 1972).
Schwartz et at (U.S. Pat. No. 5,420,285; U.S. Pat. No. 5,753,520; U.S. Pat. No. 5,420,285; J. Nucl. Med. 31(12):2022, 1990 and Bioconjug. Chem. 2(5):333, 1991) describe the preparation of succinimidyl 6-hydraziniumnicotinate hydrochloride for the one-step modification of amino groups on proteins and other molecules to incorporate pyridylhydrazine moieties on proteins for the specific purpose of binding technetium-99m for in vivo diagnostic purpose. Subsequently Schwartz (U.S. patent application Ser. No. 09/630,060) describe novel oligonucleotide aldehyde and hydrazine phosphoramidite reagents for incorporation of aldehydes and hydrazines on synthetic oligonucleotides including aromatic and heteroaromatic aldehydes and hydrazines. Triphosphates incorporating both aromatic hydrazine and aromatic aldehydes have been described by Schwartz and Hogrefe (U.S. Pat. No. 6,686,461).
Cytidine and deoxycytidine moieties in polynucleotides can be transformed into 4-N-aminocytidine (4-hyd-C), an aromatic hydrazine, by treatment with hydrazine/bisulfite at neutral pH. Nitta et al. Eur. J. Biochem. 157(2):427, 1986 has described crosslinking between 16S ribosomal RNA and protein S4 in E. coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate. Also Musso et al., (U.S. Pat. No. 5,130,446) describe labeling of 4-N-aminocytidine moieties on hydrazine/bisulfite treated DNA to yield a fluorescently labeled polynucleotide. Bittner et al. (U.S. Pat. No. 5,491,224) also describe the labeling of transaminated DNA with fluorescent moieties possessing moieties that react with the transaminated cytosine such as fluorophores possessing succinimidyl esters.
In all of the aforementioned references the biomolecule is fluorescently labeled with a fluorescent molecule. Unfortunately as previously stated the processes or methods used to prepare the conjugate can often times cause quenching or photobleaching of the fluorophore. In addition, during use the unbound fluorescently labeled conjugate must be removed to obtain an accurate fluorescent signal.
Therefore, there is a need in the field for a fluorescent label that is resistant to reaction conditions necessary for producing a labeled biomolecule and does not require removal of the unbound fluorescently labeled biomolecule from the detection reaction mixture to obtain a accurate and/or quantitative signal. There is also a need for fluorophores that may be formed under standard assay conditions from pro-fluorophores which, are stable under various laboratory conditions and by a reaction that is highly specific and efficient.
To date the most commonly used method to link, immobilize and detect biomolecules is the biotin/streptavidin ligand/receptor couple. Biotin (FIG. 1) is a small molecule, MW 250, that binds to streptavidin with an association constant of 1015. The extremely high binding constant and fast kinetics of binding and the stability of avidin under a variety of conditions make this an ideal ligand/receptor pair for these purposes. Biotin has been modified to include amino, thiol and carbohydrate reactive moieties, i.e. succinimidyl ester, maleimido and hydrazide respectively, to allow easy incorporation into a large variety of biomolecules. To accomplish detection of an analyte, biotin is conjugated to a probing biomolecule such as an antibody or an oligonucleotide. Following binding of the biotinylated biomolecule to its receptor or complement, an avidin/reporter conjugate such as an avidin/fluorophore conjugate or a avidin/reporter enzyme conjugate is added and allowed to bind to biotinylated probe and visualized by fluorescence detection or addition of a substrate that emits light or precipitates a colored insoluble product on enzymatic processing (Heitzmann H., Richards F. M., Proc. Natl. Acad. Sci. USA 71:3537-3541, 1974; Diamandis E. P., Christopoulos T. K., Clin. Chem. 37:625-636, 1991; Wilchek M. Methods Enzymol Vol. 184, 1990; Savage, M. D. et al., 1992 Avidin-Biotin Chemistry: A Handbook. Rockford, Ill.: Pierce Chemical Co.).
Following conjugation it is important to determine that the probe molecule has been biotinylated and to quantify the number of biotins now conjugated to the probe molecule. To this end two multi-step indirect assays have been developed. The first assay is the HABA ([2-(4′-hydroxyazobenzene)] benzoic acid) assay developed by Green (Green, N. M. Biochem. J., 94, 23c-24, 1965). To quantify biotin label incorporation, a solution containing the biotinylated protein is added to a mixture of HABA and avidin. Because of its higher affinity for avidin, biotin displaces the HABA from its interaction with avidin and the absorption at 500 nm decreases proportionately. By this method, an unknown amount of biotin present in a solution can be evaluated in a single cuvette by measuring the absorbance of the HABA-avidin solution before and after addition of the biotin-containing sample. The change in absorbance relates to the amount of biotin in the sample.
The second more sensitive fluorescence-based multi-step assay developed by Molecular Probes (recently acquired by Invitrogen Corporation in Carlsbad, Calif.) is the ‘Fluoreporter Biotin Quantitation Assay’ that is based on fluorescence resonance energy transfer (FRET) quenching wherein an avidin molecule is labeled with a fluorophore and its binding sites are occupied with a fluorescent molecule that quenches the covalently linked fluorophore until the quencher in the binding site is displaced by a higher binding biotin molecule resulting in fluorescence of the covalently attached fluorophore. While this assay is sensitive to 50-100 ρmol range it requires many processing steps and a fluorimeter or multi-well fluorimeter. It is also recommended to digest the biotinylated protein prior to the assay to expose any sterically encumbered biotins.
Consequently there is a need in the field for a assay wherein the number of biotins covalently linked to a biomolecule could be determined by direct methods such as spectroscopic means.